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2005 33: 726Toxicol PatholDominguez, Gail E. Sonenshein, Robert D. Cardiff, Zhi-Xiong Jim Xiao, David H. Sherr and David C. Seldin

Nicolas Currier, Sandra E. Solomon, Elizabeth G. Demicco, Donny L. F. Chang, Marganit Farago, Haoqiang Ying, IsabelOncogenic Signaling Pathways Activated in DMBA-Induced Mouse Mammary Tumors

  

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Toxicologic Pathology, 33:726–737, 2005Copyright C© by the Society of Toxicologic PathologyISSN: 0192-6233 print / 1533-1601 onlineDOI: 10.1080/01926230500352226

Oncogenic Signaling Pathways Activated in DMBA-Induced MouseMammary Tumors

NICOLAS CURRIER,1,4 SANDRA E. SOLOMON,3,4 ELIZABETH G. DEMICCO,2,4 DONNY L. F. CHANG,2,4

MARGANIT FARAGO,1,4 HAOQIANG YING,2,4 ISABEL DOMINGUEZ,1,4 GAIL E. SONENSHEIN,2,4 ROBERT D. CARDIFF,5ZHI-XIONG JIM XIAO,2,4 DAVID H. SHERR,3,4 AND DAVID C. SELDIN1,4

1Boston University School of Medicine, Department of Medicine, Boston, Massachusetts, USA2Boston University School of Medicine, Department of Biochemistry, Boston, Massachusetts, USA

3Boston University School of Public Health, Department of Environmental Health, Boston, Massachusetts, USA4Boston Medical Center, Women’s Health Interdisciplinary Research Center, Boston, Massachusetts, USA

5University of California-Davis, Center for Comparative Medicine, Davis, California, USA

ABSTRACT

Only about 5% of human breast cancers can be attributed to inheritance of breast cancer susceptibility genes, while the balance are consideredto be sporadic in origin. Breast cancer incidence varies with diet and other environmental influences, including carcinogen exposure. However, theeffects of environmental carcinogens on cell growth control pathways are poorly understood. Here we have examined oncogenic signaling pathwaysthat are activated in mammary tumors in mice treated with the prototypical polycyclic aromatic hydrocarbon (PAH) 7,12-dimethylbenz[a]anthracene(DMBA). In female FVB mice given 6 doses of 1 mg of DMBA by weekly gavage beginning at 5 weeks of age, all of the mice developed tumorsby 34 weeks of age (median 20 weeks after beginning DMBA); 75% of the mice had mammary tumors. DMBA-induced mammary tumors exhibitedelevated expression of the aryl hydrocarbon receptor (AhR), c-myc, cyclin D1, and hyperphosphorylated retinoblastoma (Rb) protein. Because ofthis, the activation of upstream regulatory pathways was assessed, and elements of the Wnt signaling pathway, the NF-κB pathway, and the prolylisomerase Pin-1 were found to be frequently up-regulated in the tumors when compared to normal mammary gland controls. These data suggest thatenvironmental carcinogens can produce long-lasting alterations in growth and anti-apoptotic pathways, leading to mammary tumorigenesis.

Keywords. DMBA; Wnt; NF-κB; AhR; Pin-1; mammary tumorigenesis.

INTRODUCTION

Although the second leading cause of cancer mortality inwomen is breast cancer, we still do not have a very com-plete understanding of the mechanisms of mammary tumori-genesis. Extensive investigation has identified breast cancersusceptibility genes that can be inherited, but these appearto play a minor role in most cases of breast cancer. Thegeographic variation in incidence of breast cancer suggeststhat environmental and dietary factors play a significant role.A major class of environmental carcinogens is a family ofstructurally related chemicals, the polycyclic aromatic hydro-carbons (PAH). These carcinogens and related halogenatedcompounds have been implicated in mammary tumorigene-sis by epidemiological and laboratory studies. DMBA (7,12-dimethylbenz[a]anthracene) is a prototypical PAH that hasbeen used to promote tumors in laboratory animals (Medina,1974). Mammary tumors can be produced in rodents fol-lowing administration of DMBA by oral gavage, leading toup-regulation of the cellular cytosolic receptor for DMBA,the aryl hydrocarbon receptor (AhR) (Trombino et al., 2000).

Upon ligand activation, the AhR translocates into the nu-cleus and associates with the cofactor ARNT, the AhR nucleartranslocation protein (Denison and Nagy, 2003; Swansonet al., 1995). This activated AhR/ARNT complex then bindsto specific DNA recognition sites upstream of AhR re-

Address correspondence to: David C. Seldin, 650 Albany Street, Boston,Massachusetts 02118; e-mail: dseldin@bumc.bu.edu

sponsive genes and induces gene transcription (Denisonand Nagy, 2003). The early steps in tumorigenesis involveAhR-dependent up-regulation of cytochrome P450 enzymes,which metabolize DMBA into a mutagenic epoxide interme-diate that readily forms DNA adducts. These adducts are asso-ciated with DNA mutations and the malignant transformationthat is thought to be involved in PAH-mediated carcinogene-sis (Nebert et al., 1990; Rundle et al., 2000). However, thereis growing evidence that the AhR also regulates cell growth(Abdelrahim et al., 2003; Ge and Elferink, 1998; Holcomband Safe, 1994; Ma and Whitlock, 1996; Puga et al., 2000)and survival (Caruso et al., 2004; Matikainen et al., 2001),suggesting that there are additional mechanisms throughwhich the AhR may contribute to mammary tumorigenesis.Furthermore, while PAH-mediated DNA damage may be animportant initiation factor, it has also been suggested thatDNA damage may not be sufficient for the development oftumors (Miller and Miller, 1981).

We hypothesized that epigenetic changes in oncogene ex-pression and intracellular signaling may play important roles.To test whether additional biochemical changes may occurthat could lead to mammary tumor formation after PAH expo-sure, we established an oral dose regimen of DMBA that re-sulted in mammary tumors in 75% of FVB mice by 30 weeksof age, associated with tonic up-regulation of the AhR. RNAtranscripts for cyclin D1 and c-myc, important oncogenes inhuman breast cancer (Bartkova et al., 1994; Jamerson et al.,2004), were increased in many of the DMBA-induced mam-mary tumors. These results led us to examine the effects of

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DMBA treatment on upstream regulators of c-myc and cyclinD1, including the NF-κB and Wnt signal transduction path-ways, and the prolyl isomerase Pin1, an important integratorof multiple signaling pathways.

NF-κB/Rel is a structurally and evolutionary conservedfamily of transcription factors. NF-κB is an important regula-tor of mammary gland development, where it controls prolif-eration and branching (Brantley et al., 2001; Cao et al., 2001),and also protects the epithelium during apoptotic alveolar in-volution (Clarkson et al., 2000). We and others have demon-strated constitutive activation of NF-κB factors in breast can-cer: high levels of nuclear NF-κB were found in the majorityof primary human breast and DMBA-induced rat mammarytumor specimens, breast cancer cell lines, and carcinogen-transformed human mammary epithelial cells (Sovak et al.,1997). NF-κB up-regulation can precede carcinogen-inducedrat mammary tumor formation (Kim et al., 2000), and overex-pression of the NF-κB family member c-Rel in the mammarygland promotes breast cancer in transgenic mice (Romieu-Mourez et al., 2003). NF-κB can activate transcription ofcyclin D1 (Joyce et al., 2001), and NF-κB and AhR act coop-eratively to transactivate the murine proto-oncogene c-myc(Kim et al., 2000).

Another pathway that has been implicated in carcinogen-induced tumorigenesis, and may also lead to transactivationof the c-myc and cyclin D1 oncogenes is the Wnt pathway.The Wnt pathway was originally discovered in Drosophila asan important signaling pathway in development (Sharma andChopra, 1976). Later it was appreciated to have a role in tu-mor development (Nusse and Varmus, 1982; Rijsewijk et al.,1987). The discovery of mutations in the adenomatous poly-posis coli (APC) gene, an important tumor suppressor pro-tein in the canonical Wnt pathway, as the underlying cause ofthe familial adenomatous polyposis colon cancer syndrome,provided molecular evidence for the role of Wnt signalingin human tumors (Nagase and Nakamura, 1993). Wnts are afamily of secreted glycoproteins that bind to Frizzled (Fz)7 transmembrane cell surface receptors. Activation of Fzleads to inhibition of glycogen synthase kinase 3β (GSK3β)via the Disheveled (Dvl) protein. Inhibition of GSK3β pre-vents N-terminal phosphorylation, and subsequent ubiquiti-nation and degradation of β-catenin. The failure to degradeβ-catenin then leads to cytoplasmic accumulation and nu-clear translocation. Once in the nucleus, β-catenin binds tran-scription factors of the T cell (TCF) and lymphoid enhancing(LEF) families and stimulates transactivation of various genessuch as cyclin D1 and c-myc, which are believed to be cen-tral to the tumorigenic potential of Wnt signaling (Polakis,2000).

Cyclin D1 promotes cell growth by activating cyclin-dependent kinases (Cdks) 4 and 6, which phosphorylate theretinoblastoma protein (Rb), a critical step in regulation ofthe restriction point at the G1/S transition (Weinberg, 1995).Recent work from our lab has identified CK2, a ubiqui-tously expressed serine/threonine kinase consisting of 2 αor α′ catalytic subunits and 2 regulatory β subunits, as apositive regulator of β-catenin stability and Wnt signalingin both mammalian cells (Song et al., 2003) and in Xeno-pus embryos, where it has Wnt-like axis determining activ-ity (Dominguez et al., 2004). We and others have demon-strated that constitutive activation of canonical Wnt signaling

(through overexpression of CK2α (Landesman-Bollag et al.,2001) kinase inactive-GSK (Farrago et al., Cancer Research,in press), β-catenin (Michaelson and Leder, 2001) or Wnts(Li et al., 2000)) leads to mammary tumorigenesis, and squa-mous metaplasia (Miyoshi et al., 2002) in mouse models.Moreover, aberrant constitutive activation of canonical Wntsignaling has been frequently found in primary human breasttumors and in human breast cancer cell lines (Brown, 2001;Smalley and Dale, 2001). Activation of the Wnt pathway hasbeen frequently seen in carcinogen-induced tumors, partic-ularly those of the gastrointestinal tract in rodents (Koesterset al., 2001; Nozaki et al., 2003; Takahashi et al., 1998;Takahashi and Wakabayashi, 2004; Ubagai et al., 2002); how-ever, there are few studies of the contribution of Wnt to mam-mary tumorigenesis. In a recent paper, the frequency of Wntpathway mutations induced in mice by 1,3-butadiene wasonly 18% (Zhuang et al., 2002).

The prolyl isomerase Pin1, also shown to be up-regulatedin human mammary tumors, has also been implicated in ac-tivation of Wnt signaling through various mechanisms. Pin1is a prolyl isomerase that catalyzes the cis-trans isomeriza-tion of pSer/Thr-Pro motifs, and thereby facilitates conforma-tional changes that affect a wide variety of protein functionsincluding subcellular localization, catalytic activity, protein-protein interactions, and protein turnover. Up-regulation ofPin1 has been observed in a variety of human cancers (Baoet al., 2004), including breast cancer, where a strong corre-lation has been observed between Pin-1 and β-catenin up-regulation (Ryo et al., 2001). Pin1 directly potentiates Wntsignaling and expression of the target genes cyclin-D1 andc-myc by facilitating β-catenin release from APC, the adeno-matous polyposis coli gene product (Ryo et al., 2001). Pin-1also directly binds to and stabilizes cyclin D1, and Pin-1knockout mice demonstrate many of the same phenotypes ascyclin D1 null mice, i.e., impairment of normal mammarygland development and retinal hypoplasia (Liou et al., 2002).

To begin to elucidate the mechanism leading to the DMBA-induced increase in cyclin D1 and c-myc, we assessed path-ways implicated in the regulation of these genes by comparingtumor samples to normal mammary glands. Here, we demon-strate activation of signaling through the AhR, NF-κB, theWnt signaling pathway and Pin-1.

MATERIALS AND METHODS

DMBA TreatmentVirgin female FVB/N mice were treated according to a

protocol approved by the Boston University Institutional An-imal Care and Use Committee. Mice were housed in a 2-waybarrier at the Boston University School of Medicine animalfacility in accordance with the regulations of the AmericanAssociation for the Accreditation of Laboratory Animal Care.Twenty mice were each given 6 weekly 1.0 mg doses ofDMBA in 0.2 ml of sesame oil by oral gavage, beginningat 5 weeks of age. Mice were then mated continuously toprovide an oscillating hormonal environment and followeduntil either tumors developed or the mice died. Mice bear-ing tumors >0.5 cm were euthanized by CO2 inhalation andnecropsied. Mammary tumors and grossly normal mammaryglands from parous age-matched control FVB mice wereexcised and portions of the tissues were prepared for

728 CURRIER ET AL. TOXICOLOGIC PATHOLOGY

histology. The remaining tissue was frozen on dry ice formolecular and biochemical studies. Frozen tissues werestored at −80◦C.

HistologyUpon necropsy, tumors and organs were removed and

immediately fixed in Optimal Fix (American HistologyReagent Company, Inc). The tissues were processed, em-bedded in paraffin, and sectioned at 7 µ. The sectionswere mounted on glass slides and stained with hema-toxylin and eosin using routine laboratory procedures in theMutant Mouse Pathology Laboratory at the University ofCalifornia, Davis. Sections were compared with other spec-imens in the extensive mouse mammary tumor database at〈http://imagearchive.compmed.ucdavis.edu〉.Immunoblot Analysis

Whole cell protein extracts were prepared by homog-enizing frozen tumors or mammary gland specimens inlysis buffer containing a cocktail of protease inhibitors(50 mM Tris-HCl pH 8.0, 1% Nonidet P-40, 125 mM NaCl,1 mM NaF, 1mM phenylmethylsulfonyl fluoride (PMSF),1 ug/ml aprotinin, 1 ug/ml pepstatin, 1 ug/ml leupeptin, 1 mMNa3VO4, and 10 mM sodium pyrophosphate) (see EMSAmethods for nuclear extraction methods). Equal amountsof protein as determined by BCA protein assay (Pierce,Rockford, IL) were diluted with 4X sample loading buffer(100 mM Tris-HCl, pH 6.8, 200 mM dithiothretol, 4% SDS,0.2% bromophenol blue, 20% glycerol), boiled, and loadedonto 8% polyacrylamide gels.

Electrophoresis was performed in a Bio-Rad Mini Pro-tean II gel system at 110V for 2 hours. After electrophore-sis, gels were transferred onto PVDF membranes (MilliporeInc.). Membranes were blocked in 5% milk in 1X TBS,0.05% Tween, incubated with an appropriate primary anti-body, washed, incubated with HRP-conjugated secondary an-tibody (Santa Cruz Biotechnology, Santa Cruz, CA), washedagain and visualized by ECL (Pierce, Rockford, IL) accordingto the manufacturer’s instructions. Primary antibodies werethe following antibodies: anti-β-catenin (BD, Upstate), anti-β-actin (SIGMA or C-11 Santa Cruz), anti-CK2α (BD), anti-GSK3β (Stressgen), anti-AhR (BioMol), Pin1 (Ab-1, Onco-gene), Phospho-specific Rb (p-780, p-795, p-807/811, CellSignaling), pan-Rb antibody (G3-245, BD Pharmingen). Forquantitative analysis of each band, integrated pixel densityminus background density was determined using a Fluor-SMultiImager and analysis was done using Quantity One soft-ware (Bio-Rad, Hercules, CA).

RNA Extraction and Reverse TranscriptionFor expression analysis, total RNA was extracted from

mouse tissues by homogenization of frozen tissue in 4MGuanidine Hydrochloride (American Bioanalytical), fol-lowed by ultracentrifugation (16 hours, 42,000 rpm) in a5.7 M cesium chloride (American Bioanalytical), 0.025 MNaOAc (American Bioanalytical) gradient. After DNasetreatment (Roche, Indianapolis, IN), RNA was reextractedusing buffered phenol/choloform (American Bioanalytical),and ethanol precipitated. One µg RNA was then reversetranscribed using the ProSTAR First Strand RT-PCR kit(Stratagene, La Jolla, CA).

Quantitative Real-Time PCR (QPCR)Twenty-five µl reactions were prepared by mixing 8 ng of

cDNA, 12.5 µl of Taqman Universal PCR Mastermix (Ap-plied Biosystems, Foster City, CA), and 1.25 µl of an As-say on Demand Gene Expression Reagent (Applied Biosys-tems, Foster City, CA) for the gene of interest. These includecyclin D1 (CCND1), β-catenin (CATNB), c-myc (MYC),aryl-hydrocarbon receptor (AHR), or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an endogenous con-trol. QPCR was performed in an ABI Prism 7000 SequenceDetection System (Applied Biosystems, Foster City, CA).The initial step was for 10 min at 95◦C followed by 40 cy-cles (95◦C for 15 seconds, 60◦C for 60 seconds). Backgroundsignal was eliminated and Ct values were determined usingthe SDS version 1.1 analysis software (Applied Biosystems,Foster City, CA). Standard curves within the linear range forGAPDH and gene of interest were performed.

Electromobility Shift Assay (EMSA) and Nuclear ExtractionFrozen tumor tissue was pulverized in liquid nitrogen with

a mortar and pestle and resuspended at a concentration of1 g/ml in homogenization buffer (10 mM Hepes pH 7.9,10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 50 mM sucrose,1 mM DTT, 0.5 mM PMSF, 5 µg/ml leupeptin, and 5 µg/mlaprotinin, Sigma Chemical Co., St. Louis, MO). Sampleswere Dounce-homogenized for 20 strokes with a loose pestleand then 20 strokes with a tight pestle. The KCl concentra-tion was then adjusted to 100 mM and the nuclei were washedtwice. Nuclear proteins were extracted on ice for 30 minutesin 2 packed nuclear volumes of 10 mM Hepes pH 7.9, 400 mMNaCl, 0.1 mM EDTA, 0.1 mM EGTA, 20% glycerol, 1 mMDTT, 0.5 mM PMSF, 0.5 µg/ml leupeptin, and 5 µg/ml apro-tinin. Protein concentration was determined using the Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA). Thesequence of the upstream regulatory element (URE) NF-κB-binding oligonucleotide from the c-myc gene is as follows: 5′-GATCCAAGTCCGGGTTTTCCCCAACC-3′ (Duyao et al.,1990), where the core element is underlined. The Octomer-1(Oct-1) binding oligonucleotide has the following sequence:5′-TGTCGAATGCAAATCACTAGAA-3′. Nuclear extractssamples (5 µg) were subjected to EMSA, as described (Sovaket al., 1997).

StatisticsFor all quantifiable measurements, p-values were obtained

using the Student’s t-test comparing values from the tumorset versus values from the set of normal samples, using thestatistical analysis package in Excel.

RESULTS

Tumor-Incidence and Histology in FVB/N MiceTreated with DMBA

Female mice were treated with 6 weekly 1 mg doses ofDMBA by oral gavage beginning at 5 weeks of age. Be-ginning 4 weeks after the final dose, at 15 weeks of age,mice began to develop evidence of tumors (Figure 1, Kaplan-Meier survival plot) and were then euthanized for analysis.By 34 weeks of age (29 weeks after beginning DMBA treat-ment), all mice had developed tumors. 75% of the mice hadmammary tumors, 15% of the mice had lung tumors, 10%

Vol. 33, No. 6, 2005 SIGNALING PATHWAYS IN DMBA-INDUCED MOUSE MAMMARY TUMORS 729

FIGURE 1.—Kaplan-Meier survival plot of 20 female FVB mice dosed 6times with 1 mg DMBA by oral gavage. Deaths were scored when mice wereeuthanized because of tumors or illness, or were found dead.

had lymphomas, and 5% had skin cancers; some mice hadmore than 1 malignancy. In addition, 30% of the mice hadmyeloid hyperplasia.

These findings can be compared with large reported seriesof female FVB mice, in which 15% of mice develop bron-choalveolar lung adenomas and carcinomas by 14 m of age;lymphomas (in 6%) and skin tumors (in 3%) have been re-ported at 24 m of age (Mahler et al., 1996). Mammary glandhyperplasia and carcinomas (both associated with and inde-pendent of pituitary hyperplasia) also can occur, but havenot been reported in the first year of life (Nieto et al., 2003;Wakefield et al., 2003). The most common histologies ofthe DMBA-induced mammary tumors were squamous oradenosquamous carcinomas, accounting for 85% of the mam-mary tumors, with other tumors being of the Wnt type (Rosneret al., 2002), or scirrhous tubular, spindle cell, or papillarycarcinomas (Table 1 and Figure 2).

DMBA-Induced Mammary Tumors Have IncreasedExpression of AhR mRNA, Protein

If the AhR plays an ongoing role in mammary tumorige-nesis, we predicted it would be up-regulated in carcinogen-induced tumors. To test this hypothesis, expression levels ofthe AhR protein and mRNA transcript were measured by im-munoblot and quantitative PCR analysis, respectively. Pro-teins were extracted from tumors arising in DMBA-treatedmice and the normal mammary glands from age-matchedcontrols, and immunoblotting was performed for AhR. Theblots were stripped and re-probed for β-actin to control for

TABLE 1.—Histology of mammary tumors.

Histology No. (%)

Squamous 10 (65%)Adenosquamous 4 (20%)Wnt type 1 (5%)Scirrhous tubular carcinoma 1 (5%)Spindle cell tumor 2 (10%)Papillary carcinoma 1 (5%)

protein loading. AhR protein expression in 7 of 11 mammarytumors was considerably higher than in the controls (Fig-ure 3a, 3b). The bands were quantitated by phosphoimageranalysis, and the average level of AhR protein (expressed as anormalized ratio of Fluor-S units compared to actin) in tumorswas 0.98 ± 0.09 (mean ± S.E.) as compared to 0.08 ± 0.03in the controls (p = 0.011). Similar results were obtained in4 separate experiments using 2 different AhR antibodies.

To measure the level of AhR mRNA, quantitative PCR(QPCR) was performed in tumor and normal samples, withGAPDH expression used for normalization. The ratio of AhRmRNA in each individual sample as compared to the meanof the normal tissues is presented as the fold-change. Sim-ilar to AhR protein results, AhR mRNA levels were higherin the majority of DMBA-induced tumors, as compared tonormal mammary tissue (Figure 3c), although the magnitudeof AhR mRNA and protein levels did not always directly cor-relate (e.g., Tumor T3). These results suggest that AhR up-regulation in mammary tumors may be regulated at multiplelevels, but are consistent with a role for the AhR in mammarytumorigenesis.

Cyclin D1 and c-myc mRNA Levels in DMBA-InducedMammary Tumors

Two cell cycle regulators that have been shown to be impor-tant for the progression of mammary tumorigenesis are thecyclin D1 and c-myc oncogenes. Thus, in our initial studies,quantitative PCR was performed to compare mRNA levelsof these genes in tumor and normal samples. The ratio of cy-clin D1 mRNA in each individual sample as compared to themean of the normal tissues is presented as the fold-change,normalized to GAPDH expression (Figure 4a). Six out of12 tumor samples tested showed increased cyclin D1 mRNAlevels, which represents a statistically significant increase inthe average cyclin D1 mRNA in the tumors versus controls[9.30 ± 1.27 vs. 1.00 ± 0.00 (p = 0.086)]. In contrast tocyclin D1, an increase in c-myc mRNA was observed only ina few of the tumors (Figure 4b).

Hyperphosphorylated Rb Protein Expressionin DMBA-Induced Mammary Tumors

G1 kinases, including cyclin D1-associated kinases, di-rectly phosphorylate Rb, and the hyperphosphorylated Rbdissociates from E2F, which is critical in promoting cell cy-cle progression (Weinberg, 1995). Thus, Rb hyperphospho-rylation is a measure of inactivation of Rb-mediated growthsuppression and deregulated cell cycle control. Therefore, weexamined the phosphorylation status of Rb protein in DMBA-induced mammary tumors. Increased amounts of phosphoRband an increased proportion of Rb in the phosphorylated formwas readily detected in DMBA tumors, in contrast to normalmammary gland (Figure 5a, 5b). Hyperphosphorylated Rbprotein levels in tumors were 0.34 ± 0.03 compared with0.03 ± 0.01 in normal controls (p = 0.013), indicating thatRb is inactivated in DMBA-induced mammary tumors.

DMBA-Induced Mammary Tumors Exhibit IncreasedNuclear NF-κB Binding

The NF-κB pathway is capable of regulating transcrip-tion of the c-myc and cyclin D1 genes. To determine whetherDMBA-induced mouse mammary tumors have constitutively

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FIGURE 2.—Histology of FVB mouse mammary tumors occuring after DMBA exposure. (A) Branched ductal dysmorphogenesis in a Wnt-type tumor withextensive fibrosis; (B) A tubulo-papillary carcinoma; (C) Tumor with features of EMT (epithelial to mesenchymal transition); (D) Adenosquamous carcinoma;(E) Squamous carcinoma; (F) Mixed tumor, Dunn type B also with EMT.

active nuclear-NF-κB, EMSAs were performed using the c-myc promoter URE as a probe (Duyao et al., 1990). Nu-clear extracts from the DMBA-induced mouse mammary tu-mors were compared with those from mammary glands ofage-matched female FVB/N mice or from a nulliparous ani-mal (Figure 6). NF-κB bands were identified using supershiftanalysis (data not shown). While NF-κB binding in normalmammary gland fluctuates with the estrus cycle, the predom-inant complex seen was made up of p50 homodimers (Band5) and a minor amount of p50/p65 heterodimers (Band 1),as seen previously (Clarkson et al., 2002). In addition, someclipping of p50 occurred (Band 6), as we have observed pre-viously (Pianetti et al., 2001). In contrast, DMBA-inducedtumors displayed increased levels of the potent transacti-vating complexes p50/p65, p50/c-Rel (Band 2), p52/RelB(Band 3), in addition to p50 or p52 (Band 2) homodimers.Equal loading and protein integrity of the nuclear extractswas confirmed by analysis of Oct-1 binding. Thus, DMBA-induced mammary tumors are characterized by elevated lev-

els of NF-κB binding complexes known to be able to activatetranscription.

DMBA-Induced Mammary Tumors Have ElevatedExpression of Canonical Wnt Signaling Components

To evaluate the Wnt signaling pathway in DMBA-inducedmammary tumors, we initially measured whole cell and nu-clear levels of β-catenin protein, the essential transcriptionalcofactor for canonical Wnt signaling. In the whole cell ex-tracts, substantial up-regulation ofβ-catenin was found in 6 of12 tumor samples tested, when compared to mammary glandsfrom untreated, age-matched FVB mice (Figure 7a, 7b). Theaverage level of β-catenin, expressed as a normalized ratio ofFluor-S units compared to β-actin, was significantly higherin the tumors, as compared to the normal controls [0.36 ±0.35 vs. 0.03 ± 0.01 (p = 0.008)]. Similar results were ob-tained in 3 separate experiments using 2 different β-cateninantibodies (data not shown). We also examined β-cateninexpression in the nuclear extracts prepared for EMSA, and

Vol. 33, No. 6, 2005 SIGNALING PATHWAYS IN DMBA-INDUCED MOUSE MAMMARY TUMORS 731

FIGURE 3.—AhR protein and mRNA are up-regulated in mammary tumors ofDMBA-treated mice. (A) Samples (100 µg) of protein from whole cell extractsof mammary tumors (T) and normal mammary glands (N) from age-matchedcontrol FVB mice were electrophoresed, transferred to PVDF membranes andprobed with an AhR specific antibody (upper panel, representative lanes froma single gel). β-actin was used as a loading control (lower panel). (B) Quanti-tative analysis was performed by phosphoimaging, and expression of AhR wasnormalized to β-actin in each sample. The graph represents the AhR levels inDMBA-induced mammary tumors (black bars) and age-matched normal mam-mary glands (white bars). mRNA was extracted from breast tumors and normalmammary glands, reverse transcribed, and subjected to QPCR. Results are ex-pressed as ratios of mRNA levels in tumors compared to normal FVB mammaryglands, each normalized for GAPDH. (C) AhR mRNA levels in DMBA-inducedmammary tumors (black bars), and normal mammary glands (white bars).

found a similar pattern of increased nuclear β-catenin (datanot shown).

One of the positive regulators of β-catenin protein sta-bility is the serine-threonine kinase CK2, which phosphory-lates β-catenin in the armadillo repeat region and stabilizes it.Interestingly, all but 1 tumor demonstrated a significant up-regulation of the major catalytic CK2α subunit (Figure 8a, 8b)

FIGURE 4.—The mRNA levels of c-myc and cyclin D1 are elevated in DMBA-induced mammary tumors. mRNA was extracted from breast tumors and normalmammary glands, reverse transcribed, and subjected to QPCR. Results are ex-pressed as ratios of mRNA levels in tumors compared to normal FVB mammaryglands, each normalized for GAPDH. (A) Cyclin D1 mRNA levels in DMBA-induced mammary tumors (black bars), and normal mammary glands (whitebars). Similar results were obtained in 3 separate experiments (data not shown);(B) c-myc mRNA levels in the indicated DMBA-induced mammary tumors(black bars), and normal mammary glands (white bars).

as compared to the normal tissues. Mean tumor and normalvalues were [0.24 ± 0.014 vs. 0.003 ± 0.001 (p = 0.001)] re-spectively. The up-regulation of CK2α correlated well withthe up-regulation of β-catenin (Figure 8c), consistent withthe putative role of CK2 as a positive Wnt regulator. Theseresults demonstrate activation of the Wnt signaling pathwayin DMBA-induced mammary tumors.

Increased Pin1 Protein Expression in DMBA-InducedMammary Tumors

Expression of the prolyl-isomerase Pin1 is often increasedin human cancers, including breast cancer (Bao et al., 2004).Therefore, we examined Pin1 expression in the DMBA-induced mammary tumors. As shown in Figure 9, Pin1 pro-tein expression was significantly increased in many of themammary tumors, compared to age-matched normal FVBmice mammary glands. The average level of Pin1, normal-ized ratio of Fluor-S units compared to β-actin, was 0.25 ±

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FIGURE 5.—DMBA-induced mammary tumors have elevated hyperphosphorylated Rb (ppRb) protein. (A) Samples (100 µg of protein) from wholecell extracts of mammary tumors (T) and normal mammary glands from age-matched control FVB mice (N) were subjected to immunoblot analyses us-ing antibodies for hyperphosphorylated Rb (ppRb), total Rb, or β-actin (C-11, Santa Cruz). (B) Quantitative analyses were performed as described inFigure 2.

0.02 in the tumors compared with 0.08 ± 0.06 in the controls(p = 0.032) (Figure 9a, 9b). Interestingly, QPCR analysisof Pin1 mRNA levels showed no significant differences be-tween tumors and the age-matched normal controls (data notshown), suggesting that elevated Pin1 expression in DMBA-induced mammary tumors is likely due to a posttranscrip-tional mechanism. A correlation between the de-regulationof Pin1 protein and several important cell cycle regulators wasobserved (Table 2). In many of the tumor samples, increasedPin1 expression correlated well with an increase β-catenin(Figure 9c), and also with the level of hyperphosphorylatedRb and cyclin D1 mRNA (Table 2).

DISCUSSION

The present study describes an oral dose regimen of the en-vironmental carcinogen DMBA capable of generating mam-mary tumors in FVB strain mice. We hypothesized that,

FIGURE 6.—DMBA-induced murine mammary tumors display high levels of NF-κB binding compared to normal mammary gland. Nuclear extracts were preparedfrom untreated normal mouse mammary glands (N) and mammary tumor tissue (T) from DMBA-treated mice, and subjected to EMSA using the URE NF-κBoligonucleotide (upper panel). Essentially equal loading was confirmed by Oct-1 binding (lower panel). Multiple NF-κB complexes were detected and these weregiven numbers from 1 to 6, and identified by supershift EMSA (see text).

in addition to its genotoxic properties, DMBA would alsoalter oncogenic signaling pathways in order to transformcells. Here we have shown evidence of up-regulation of cellcycle regulators that have well-established roles in mam-mary tumorigenesis and of the signaling pathways capable ofup-regulating them. These data provide evidence of specificbiochemical changes that accompany tumor formation inPAH-induced mammary tumors, and activation of criticalregulatory pathways that can contribute to growth and op-pose apoptosis in cancer in the process of carcinogen-inducedmammary tumorigenesis in mice. These observations expandupon previous reports of DNA damaging events such as theinduction of point mutations in genes such as c-H-ras byDMBA (Cardiff et al., 1988).

As it is believed that both genotoxic and mitogenic activi-ties of environmental carcinogens may be mediated throughthe aryl hydrocarbon receptor (AhR), our initial experiments

Vol. 33, No. 6, 2005 SIGNALING PATHWAYS IN DMBA-INDUCED MOUSE MAMMARY TUMORS 733

FIGURE 7.—β-catenin protein is up-regulated in mammary tumors of DMBA-treated mice. (A) Whole cell extracts (15 µg of protein) from mammary tumors(T) and normal mammary glands from age-matched control FVB mice (N)were subjected to immunoblotting with a β-catenin antibody (upper panels).Spleen extract (S) was blotted as a positive control, and β-actin was used asa loading control (lower panels). (B) Quantitative analysis was performed byphosphoimaging, and expression of β-catenin in the whole cell extracts wasnormalized to β-actin in the same sample in the DMBA-induced mammarytumors (black bars) and age-matched normal mammary glands (white bars).

involved measuring levels of the AhR protein and transcript.In tumors arising in the carcinogen-treated mice, as we havepreviously seen in rats treated with a single dose of DMBA(Trombino et al., 2000), there was evidence of constitutiveup-regulation of the AhR itself. Preliminary data identifiedfrequent up-regulation of an AhR target gene, CYP1B1, al-though CYP1B1 levels did not always correlate with AhRlevels in any given tumor (data not shown). Since it has beenwell established that CYP1B1 is involved in activation ofDMBA to its genotoxic metabolites and in metabolism ofestradiol into mutagenic intermediates, our data suggest thatAhR up-regulation in tumors may contribute to ongoing ge-netic changes through CYP1B1 induction.

As a means to evaluate fundamental cell cycle regulatorsknown to be important in mammary tumorigenesis, initialstudies measured mRNA levels of the cyclin D1 and c-myconcogenes, and levels of hyperphosphorylated Rb. Quanti-tative PCR results suggested that cyclin D1 mRNA is fre-quently elevated, and c-myc is occasionally elevated. Westernblot analysis demonstrated increased amounts of phosphoRband an increased proportion of Rb in the phosphorylatedform. When we examined upstream regulatory pathways,

FIGURE 8.—CK2 is up-regulated in DMBA-induced mammary tumors.(A) Samples (15 µg of protein) from whole cell extracts of mammary tumors(T) and normal mammary glands from age-matched control FVB mice (N) weresubjected to immunoblot analysis with a CK2α antibody (upper panels). Spleenextract (S) was blotted as a positive control, and β-actin as loading control(lower panels). (B) Quantitative analyses were performed as described above.(C) Graph of the correlation between normalized β-catenin protein levels (X-axis) and CK2α levels (Y-axis) in mammary tumor samples (black diamonds)and normal mammary tissue (white diamonds).

we found evidence of constitutive NF-κB and β-cateninsignaling.

The NF-κB pathway blocks apoptosis in many cancers, andwe have demonstrated that constitutive expression of c-rel inthe mammary gland of transgenic mice produces mammarytumors (Romieu-Mourez et al., 2003). In the DMBA-inducedmammary tumors, there were functional nuclear NF-κB com-plexes that included the transactivators p65, c-Rel, RelB, and

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FIGURE 9.—Increased Pin1 protein expression in DMBA-induced mammary tumors. (A) Samples (50 µg of protein) of whole cell extracts from the indicatedmammary tumors (T) and normal mammary glands from age-matched control FVB mice (N) were subjected to immunoblot analyses using an antibody specific forPin1 or β-actin. (B) Quantitative analysis was performed as described in Figure 2. (C) Graph of the correlation between normalized Pin1 protein levels (X-axis) andβ-catenin levels (Y-axis) in mammary tumor samples (black diamonds) and normal mammary tissue (white diamonds).

partners p50 and p52. The c-myc and cyclin D1 genes aretransactivated by NF-κB heterodimeric complexes composedof p65/p50 and c-Rel/p50, but not by homodimers of p50 orp52. Thus, the relative composition of subunits regulates theactivation of these genes, suggesting a possible mechanismfor the observed up-regulation of cyclin D1 and c-myc onco-genes.

Our data also provide evidence of activation the Wnt path-way in the majority of PAH-induced mammary tumors. Asecond pathway capable of c-myc and cyclin D1 transacti-

vation. Wnt signaling can be assessed by total levels of theco-transactivator β-catenin in cells and tissues, but particu-larly by measurement of nuclear levels. Both total and nuclearβ-catenin levels were elevated in most of the tumors. Mosttumors also had elevated protein levels of the positive kinaseregulator, CK2. CK2 may be contributing to the increase intumor β-catenin, as well as stabilizing other pro-oncogenicproteins. The majority of the tumors with significantly ele-vated levels of β-catenin (T1, T9, T11) also have histolog-ical characteristics associated with Wnt pathway mammary

Vol. 33, No. 6, 2005 SIGNALING PATHWAYS IN DMBA-INDUCED MOUSE MAMMARY TUMORS 735

TABLE 2.—Summary of molecular results.

Protein mRNA

Tumor # Mammary tumor histology AhR NF-κB CK2α β-cat Pin1 ppRb cyclin D1 c-myc

T1 Mixed Wnt type and squamous cellcarcinoma

+++ + +++ ++++ ++++ ++++ ++ +++T2 Spindle cell tumor ++ + +++ − +++ + ++++ −T3 Squamous cell carcinoma with

adenosquamous features++++ ++ + + ++ − − N/A

T4 Mixed cell tumor − + +++ +++ + +++ + N/AT5 Squamous cell carcinoma with liver

and lung metastases++ N/A N/A − N/A N/A + N/A

T6 Squamous cell carcinoma + N/A − − ++ − ++ −T7 Squamous cell carcinoma + + ++++ +++ + +++ − −T8 Squamous cell carcinoma N/A +++ ++++ − − + ++ N/AT9 Adenosquamous carcinoma ++ + ++++ ++++ ++++ ++++ ++ +T10 Squamous cell carcinoma ++ ++ ++ ++ +++ ++ − −T11 Scirrhous tubular carcinoma and

adenosquamous carcinoma++ + ++ +++ + + − −

T12 Mixed squamous cell andadenosquamous carcinoma;squamous cyst

− + N/A − − − ++ N/A

tumors (Rosner et al., 2002). Additionally, squamous cellcarcinoma, seen histologically in the majority of the DMBA-induced mammary tumors, has been associated with Wnt-related tumors of the mammary gland (Rosner et al., 2002).

DMBA-induced mammary tumors also expressed elevatedlevels of Pin1, a prolyl isomerase that has been shown tobe up-regulated in a variety of human cancers, and to func-tion as a signaling integrator in the regulation of β-catenin(Pang et al., 2004; Ryo et al., 2001), cyclin D1 (Liou et al.,2002; Wulf et al., 2001), c-myc (Yeh et al., 2004), and NF-κB (Ryo et al., 2003). Consistent with these observations, aconcurrent increase in Pin1, β-catenin, and Rb hyperphos-phorylation was seen in many of the DMBA-induced mam-mary tumors (Table 2). Interestingly, while elevated cyclinD1 correlated well with hyperphosphorylated Rb in sometumors, in others it did not, suggesting that there may be ac-tivation of other non-cyclin D1-dependent Rb kinases or in-hibition of Rb phosphatases in some of the DMBA-inducedtumors. While previous studies have demonstrated an inter-action between the AhR and Rb (Ge and Elferink, 1998; Pugaet al., 2000) and shown that dioxin-induced cell cycle arrestmay occur through an AhR-mediated down-regulation of cy-clin D1 and concomitant increases in hypophosphorylated Rb(Barnes-Ellerbe et al., 2004), ours is the first to demonstratein vivo up-regulation of these 3 factors following carcino-gen treatment and suggests that in this system the AhR maybe stimulating the cell cycle. This is consistent with in vitroobservations made of mammary epithelial cells transfectedwith the AhR (Brooks and Eltom, 2005).

Our observation of activation of these known oncogenicpathways not only correlates well with previous data fromour lab using Sprague–Dawley rats (Landesman-Bollag et al.,2001; Sovak et al., 1997; Trombino et al., 2000), but withother studies of carcinogen-induced mammary tumors inwhich gene expression patterns were analyzed by microar-ray. In particular, various studies describe up-regulation ofcyclin D1 transcripts in DMBA and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) induced mammary tu-mors (Kuramoto et al., 2002; Shan et al., 2002). QPCR anal-ysis of Pin1 mRNA levels showed no significant differencebetween tumors and the age-matched normal controls (data

not shown), suggesting that Pin1 up-regulation in DMBA-induced mammary tumors is likely due to a posttranscrip-tional mechanism. This observation is consistent with thelack of Pin-1 mRNA up-regulation identified by microarray.Posttranscriptional mechanisms may also contribute to theactivation of β-catenin and NF-κB.

While the current studies do not determine whether thesignaling cascades are activated sequentially or in parallelin the course of transformation, this can be tested in the fu-ture using transgenic mouse models. For instance, by treatingAhR-null mice with DMBA by this protocol, we can assessthe role of this receptor in the activation of the downstreammediators. Understanding the interaction of these signalingpathways may provide targets for novel therapeutics in thefuture. Furthermore, since many transgenic models exist inthe FVB background in which oncogenes produce mammarytumorigenesis in a multistep stochastic fashion, these stud-ies provide us with the capability of examining the inter-action of carcinogens and genetic predisposition to breastcancer.

ACKNOWLEDGMENTS

We would like to thank Craig Lenz and Dr. Shi Yang fortheir assistance in dosing the FVB mice. We would like tothank Benjamin Schlechter and Dr. Mary Williams for theirassistance with the quantitative PCR and Dr. Adrianne Rogersfor many helpful discussions. This work was supported byP01 ES11624.

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